The long-term aim of the research described in this proposal is to understand the structural basis underlying the unique substrate specificities of the human cytochrome P450 family 4 proteins, such that predictions can be made concerning the range of metabolic bioactivation reactions which each isoform catalyzes. CYP4A proteins catalyze the thermodynamically disfavored omega-hydroxylation of medium-long chain fatty acids, the rate-limiting step in dicarboxylic acid formation, whose build-up in some inborn errors of mitochondrial metabolism causes severe toxicity in humans. The CYP4B isoforms, which are the focus of the current proposal, are mainly associated with the bioactivation of a diverse range of pro-toxins, including valproic acid, carcinogenic aromatic amines and the pneumotoxic furan, 4-ipomeanol, although curiously, human CYP4B1 has been reported to lack bioactivation capacity towards several protoxins. Detailed active-site structural information with which to rationalize the discrete substrate specificities of membrane- bound CYP4B1 isoforms is not available. Therefore, the Specific Aims of the proposal are: (I) Elucidate the substrate specificities and bioactivation capacities of human holo-CYP4B1. (II) Design mechanism-based inhibitors with which to adduct and identify active-site residues of the human and rabbit orthologs. (III) Identify important amino acid contact points between CYP4B1 and its substrates by site-directed mutagenesis of the rabbit and human enzymes. These Specific aims will be accomplished by combining; (a) structure-function studies involving fatty acids, hydrocarbons, aromatic amine and furan substrates for the two species orthologs, (b) MALDI and LC/MS/MS analysis of CYP4B1 proteins adducted with acetylenic inhibitors, (c) topographical information arising from (i) the rearrangement of aryl-iron complexes formed within the active site of each protein and (ii) the magnitude of intramolecular isotope effects for CYP4B1- catalyzed benzylic hydroxylation, and (d) constructing rabbit- human CYP4B1 hybrids and applying techniques a-c above to the chimeras and relevant point mutants. Synthesis of the information derived from these complementary approaches will enable the evolution or cohesive active-site model for CYP4B1 orthologs which integrates both species differences in substrate specificity and the diverse range of bioactivatable substrates known for this enzyme.